Pollination and the subsequent invasive growth of pollen tubes into the female stigmatic and pistil tissues prior to fertilization provides an opportunity to study cell-cell interactions in flowering plants. In crucifers such as Brassica oleracea, self recognition between pollen and stigma is controlled by the multi-allelic self-incompatibility, or S locus. Pollen germination and/or tube growth are arrested at the stigma surface if pollen and stigma are borne by plants having identical S locus alleles. This arrest prevents self-fertilization and is termed the selfincompatibility (SI) response. Two related genes have been identified at the S locus using molecular methods. Of these, only one gene, the S locus glycoprotein (SLG) gene has been characterized extensively. SLG encodes a secreted glycoprotein that is highly polymorphic in different S-locus alleles, and its expression in stigmatic papillae and anthers, is consistent with models for Sl in which both pollen and stigma bear recognition determinants derived from the S locus.
Plant science has recognized for many years that hybridization of closely related plants often results in the production of an offspring (F1) generation having a combination of desirable traits previously possessed separately by the parent plants. Also, hybrid plants of various crop species commonly possess vigor or heterosis which significantly contributes to the species' crop yield. Accordingly, many hybrid crosses are of considerable economic importance.
Since plants selected for hybridization studies are commonly capable of undergoing both self-pollination (i.e., the plants are "self compatible") and crosspollination, the desired hybrid crosses have been difficult to achieve on a reliable basis while operating on a commercial scale. In order to achieve hybrid crosses on a commercial scale, it is necessary to control cross-pollination in the substantial absence of self-pollination (i.e., plants that are "self-incompatible").
In more than 3,000 species representing diverse angiosperm families, selffertilization is blocked by a genetic controlled .mechanism termed "selfincompatibility". This phenomenon facilitates out-crossing by inhibiting growth of self pollen in pistil tissue. Following self-pollination, interaction between the stigma or style and pollen elicits a defined morphological response preventing normal pollen tube growth. Fertilization is thus prevented as the pollen tubes are unable to penetrate the full length of the stigma ands-style, and gamete nuclei in the pollen are unable to reach female gametophyte tissue. The specificity of this interaction is determined by one or more genetic loci, and requires the identity of alleles carried by the male and female parents.
The control of incompatibility is attributed to a single genetic locus with multiple alleles. As with other allelomorphs, any two alleles may be carried by a diploid plant; incompatibility occurs if the alleles expressed in pollen and pistil are identical.
Incompatibility systems are generally divided into two groupings: (i) the gametophytic system in which the phenotype of a pollen grain is determined by the haploid pollen genotype; and (ii) the sporophytic system in which the pollen phenotype is determined by the genotype of the parental plant. In the sporophytic system of incompatibility, exemplified by the genus Brassica, and other crucifers belonging to the family Brassicaceae (which includes broccoli, cabbage, kale, mustards, oil seed crops and radish), the self-incompatible inhibition is localized at the stigma outer surface and occurs within minutes by failure of self-pollen to germinate and/or invade the papillar cells on the stigma surface. Thus, self-fertilization is prevented. In Brassicaceae, self-incompatibility is under control of a single multi-allelic locus (numerous alleles have been identified in natural populations), the S-locus, and phenotype of pollen is determined by diploid of the sporophyte [see Heredity 9:53 (1955)]. While the mechanism of pollen recognition is not understood, observations that self-incompatibility responses occur when the same S-allele is active in pollen and stigma suggest pollen inhibition is based on expression of a single S-locus gene in the male and female structures of the flower. Sporophytic control of pollen phenotype has furthermore been postulated to result from expression of this S-locus gene in the taperum, the layer of sporophytic cells that lines the anther and functions to nourish developing pollen grains [see Nature 218:90 (1968) and Ann Rev Plant Physio 26:403 (1975)]. Based largely on ultrastructural observations, this cellular layer is thought to act as nurse cells for developing microspores, and to synthesize and release materials that are incorporated into the outer coat (exine) of pollen grains just prior to and during tapetal dissolution late in pollen development.
A gene involved in the specific recognition of self pollen is also expected to be sporophytically expressed in the host's anthers during pollen development. While blot analysis of anther RNA has identified a low-abundance transcript with homology to the S-locus glycoprotein gene [see Ann Rev Genetics 23:121 (1989)]these studies could not distinguish between expression of the S-locus glycoprotein related genes bund in the Brassica genome.
Perhaps the most experimentally tractable route towards molecular analysis of genetic control for incompatibility is suggested by detection of antigens specific to various S-locus alleles in stigma homogenates from different Brassica strains which have been shown to correspond to glycoproteins and may be resolved in various electrophoretic systems. Several lines of evidence suggest these glycoproteins play an important role in incompatibility: (1) mobilities of these molecules vary in stigma extracts from Brassica strains with different S-locus alleles; (2) these molecules are found exclusively on the stigma surface (specifically in papillar cells), the site of the initial contact between the pollen and stigma; (3) an increased rate of synthesis of these S-locus specific glycoproteins in the developing stigma correlates well with onset of the incompatibility reaction in the stigma; and (4) glycoproteins in the stigma of self incompatible lines of B. oleracea (kale), B. campestris, and related Raphanus sativus (radish) have been identified which cosegregate with their corresponding S-alleles in the F1 and F2 generations, indicating that the gene responsible for this polymorphism is at the S-locus.
These glycoproteins have been designated S-locus specific glycoprotein ("SLSG"). Each S-genotype has its own unique glycoprotein, and to date more than twenty different SLSG have been correlated with S-alleles. Furthermore, SLSG encoded by different S alleles exhibit extensive polymorphism and are distinguishable on the basis of their characteristic charge, molecular weight and antigenic properties. Also, SLSG accumulate during stigma development and are synthesized at a maximal rate coincident with acquisition of the developing stigma for the ability to reject self pollen.
Cell-specific expression of these genes in the stigma are well characterized. S-locus gene transcripts were localized by in Situ hybridization to the papillar cells of the stigma surface [see PNAS USA 85:5551 (1988)], and S-locus gene glycoprotein products were shown by ultra structural immunocytochemistry to accumulate in the papillar cell walls [see Dev Biol 134:462 (1989)]. These results, and the genetic linkage of S-locus glycoprotein genes to the S-locus, have led to one aspect of the present invention, that is, that these genes encode the stigmatic determinants of self-incompatibility.
The S-locus glycoprotein genes, and specifically those genes from B. oleracea which have been designated as S.sub.6, S.sub.13, S.sub.14 and S.sub.22, and the S.sub.8 genome from B. campestris, are described in copending U.S. patent application Ser. No. 07/568,657, now abandoned, filed Aug. 14, 1990 which is a continuation of application 06/762,245 filed Aug. 5, 1985 and now abandoned, application 07/459,069 filed Dec. 29, 1989 and application 07/532,907 filed Jun. 4, 1990, now abandoned, the disclosures of these applications of which are incorporated in toto herein.